Conserved Transmembrane Domains Research Articles

LPXRFa and LPXRFa-R in the female swamp eel (Monopterus albus): Molecular cloning, tissue distribution and gonadal function

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide known for inhibiting gonadotropin release. In teleosts, it is also referred to as LPXRFa. While the effects of LPXRFa on gonadotropin synthesis and release, and its impact on pituitary function in teleosts have been widely studied, its specific mechanism in fish reproduction remains relatively unexplored. In this paper, LPXRFa and LPXRFa-R genes were isolated from the swamp eel brain to assess the roles of LPXRFa in the swamp eel. Their ORFs were 612 bp and 1284 bp, encoding proteins of 203 and 427 amino acids, respectively. The LPXRFa gene encodes a polypeptide possibly cleaved into three mature peptides (LPXRFa-1, −2, and −3). The LPXRFa-R receptor protein comprises seven conserved transmembrane domains. Tissue distribution analysis revealed that LPXRFa-R expression was the highest in the hypothalamus, while LPXRFa showed the highest expression in the rest of brain, with little to no expression in other tissues. The role of LPXRFa in the reproduction of the swamp eel was investigated by studying the effect of intraperitoneal injection with each of the three peptides. The intraperitoneal injection of LPXRFa-2 down-regulated serum FSH and LH levels, while that of LPXRFa-3 down-regulated serum FSH but up-regulated LH levels. Additionally, both LPXRFa-2 and LPXRFa-3 increased the expression levels of fshr, cbx3, dmrt1, gsdf, sox9al, amh, foxl2, and rab37 in the ovaries. Moreover, LPXRFa-2 also increased the ovarian expression of lhr, while LPXRFa-3 had no effect on it. Except upregulating cbx3 and foxl2 expression, LPXRFa-1 neither impacted the serum FSH and LH levels, nor did it alter the expression of other gonadal and reproductive-related genes. In summary, our findings provide the first evidence of a LPXRFa/LPXRFa-R system in the order Synbranchiformes and suggest its involvement in sex differentiation, gonadal development, and sexual reversal in the swamp eel.

Abstract P3203: Characterization Of Nrac, A Novel Cardiac And Adipose Enriched Microprotein

Objective: Nrac ( n utritionally- r egulated a dipose and c ardiac-enriched gene) was previously identified as a microprotein encoded by the long noncoding RNA A530016L24Rik (human C14orf180) with 2 conserved transmembrane domains. Nrac is highly expressed in the heart as well as in brown and white adipose tissue and its expression is decreased in response to both fasting and obesity. To date, the biological function of Nrac is unknown, however a previous study indicated it may localize to the plasma membrane in white adipocytes. The goal of this study was to examine the molecular function of Nrac in adipocytes and the heart. Methods and Results: Using a brown adipocyte cell culture system, we differentiated pre-adipocytes to mature adipocytes and analyzed Nrac expression levels by qPCR. Nrac expression was undetectable in pre-adipocytes but markedly increased during differentiation and was robustly expressed in mature adipocytes. To determine the subcellular localization of Nrac, we transfected brown pre-adipocytes with a HA-V5-eptipe tagged Nrac fusion protein (HA-V5-Nrac), differentiated these cells to mature brown adipocytes, and performed immunostaining. These experiments showed that Nrac localizes specifically to the endoplasmic reticulum, and that overexpression of Nrac led to a striking increase in intracellular lipid droplet size as determined by BODIPY staining. To analyze Nrac in vivo, we generated a novel Nrac knockout (KO) mouse line using CRISPR/Cas9. Histological analysis of brown adipose tissue (BAT) from these mice revealed that intracellular lipid droplet size was markedly decreased in Nrac KO tissue compared to BAT from WT mice. RNA-sequencing analysis of heart tissue from WT and Nrac KO mice revealed a striking downregulation of genes related to adipogenesis and fatty acid metabolism in Nrac KO hearts and an upregulation in endoplasmic reticulum stress related genes. Conclusions: Our data indicate that Nrac is an endoplasmic reticulum membrane-localized microprotein that is highly expressed in the heart and adipose tissue. Our studies indicate that Nrac is involved in adipogenesis and lipid droplet formation. Future studies will aim to define the molecular mechanisms that contribute to Nrac function in heart and adipose tissue.

Evidence of horizontal gene transfer within porB in 19 018 whole-genome Neisseria spp. isolates: a global phylogenetic analysis.

The PorB porins are the major pore-forming proteins in the genus Neisseria . The trimeric PorB porins consist of 16 highly conserved transmembrane domains that form an amphipathic β-sheet connected by short periplasmic turns and eight extracellular hydrophilic loops. These loops are immunogenic and also play an important role in mediating antimicrobial influx. This study sought to (i) characterize the variations in Neisserial loop 3(355–438 bp) associated with intermediate resistance to penicillin/tetracycline and (ii) evaluate if there was evidence of horizontal gene transfer in any of the loops. We collated an integrated database consisting of 19 018 Neisseria spp. genomes – 17 882 Neisseria gonorrhoeae , 114 Neisseria meningitidis and 1022 commensal Neisseria spp. To identify the porB alleles, a gene-by-gene approach (chewBBACA) was employed. To evaluate the presence of recombination events, the Recombination Detection Programme (RDP4) was used. In total, 3885 porB alleles were detected. Paralogues were identified in 17 Neisseria isolates. Putative recombination was identified in loop regions. Intraspecies recombination among N. gonorrhoeae isolates and interspecies recombination between N. meningitidis and commensal Neisseria spp., and N. gonorrhoeae and N. lactamica were identified. Here, we present a large-scale study of 19 018 Neisseria isolates to describe recombination and variation in the porB gene. Importantly, we found putative recombination in loop regions between the pathogenic and non-pathogenic Neisseria spp. These findings suggest the need for pheno- and genotypic surveillance of antimicrobial susceptibility in commensal Neisseria spp. to prevent the emergence of AMR in the pathogenic Neisseria . This article contains data hosted by Microreact.

Genome-wide characterization and expression analysis of the MLO gene family sheds light on powdery mildew resistance in Lagenaria siceraria

MLO (mildew locus O) genes play a vital role in plant disease defense system, especially powdery mildew (PM). Lagenaria siceraria is a distinct Cucurbitaceae crop, and PM is one of the most serious diseases threatening crop production and quality. Although MLOs have been exploited in many Cucurbitaceae species, genome-wide mining of MLO gene family in bottle gourd has not been surveyed yet. Here we identified 16 MLO genes in our recently assembled L. siceraria genome. A total of 343 unique MLO protein sequences from 20 species were characterized and compared to deduce a generally high level of purifying selection and the occurrence of regions related to candidate susceptibility factors in the evolutional divergence. LsMLOs were clustered in six clades containing seven conserved transmembrane domains and 10 clade-specific motifs along with deletion and variation. Three genes (LsMLO3, LsMLO6, and LsMLO13) in clade V showed high sequence identity with orthologues involved in PM susceptibility. The expression pattern of LsMLOs was tissue-specific but not cultivar-specific. Furthermore, it was indicated by qRT-PCR and RNA-seq that LsMLO3 and LsMLO13 were highly upregulated in response to PM stress. Subsequent sequence analysis revealed the structural deletion of LsMLO13 and a single nonsynonymous substitution of LsMLO3 in the PM-resistant genotype. Taken all together, it is speculated that LsMLO13 is likely a major PM susceptibility factor. The results of this study provide new insights into MLO family genes in bottle gourd and find a potential candidate S gene for PM tolerance breeding.

Genome-wide identification, evolution, and expression analysis of MLO gene family in melon (Cucumis melo L.).

Powdery mildew (PM) is one of the main fungal diseases that appear during the cultivation of the melon fruit crop. Mildew Resistance Locus "O" (MLO) is known as a gene family and has seven conserved transmembrane domains. An induced functional loss of a specific MLO gene could mainly confer PM resistance to melons. However, the genomic structure of MLO genes and its main role in PM resistance still remain unclear in melon. In this study, bioinformatic analysis identified a total of 14 MLO gene family members in the melon genome sequence, and these genes were distributed in an uneven manner on eight chromosomes. The phylogenetic analysis divided the CmMLO genes into five different clades, and gene structural analysis showed that genes in the same clade had similar intron and exon distribution patterns. In addition, by cloning the CmMLO gene sequence in four melon lines, analyzing the CmMLO gene expression pattern after infection, and making microscopic observations of the infection pattern of PM, we concluded that the CmMLO5 (MELO3C012438) gene plays a negative role in regulating PM-resistance in the susceptible melon line (Topmark), and the critical time point for gene function was noticed at 24 and 72 hours after PM infection. The mutational analysis exhibited a single base mutation at 572 bp, which further results in loss of protein function, thus conferring PM resistance in melon. In summary, our research evidence provides a thorough understanding of the CmMLO gene family and demonstrates their potential role in disease resistance, as well as a theoretical foundation for melon disease resistance breeding.

Genome-Wide Identification and Expression Analysis of the KCS Gene Family in Yellow Horn Reveal Their Putative Function on Abiotic Stress Responses and Wax Accumulation

The β-ketoacyl CoA synthase encoded by the KCS genes is a rate-limiting enzyme for the synthesis of very-long-chain fatty acid (VLCFA), which catalyzes the VLCFA elongation. Yellow horn (Xanthoceras sorbifolium) is a horticultural tree species known for its kernel oil, which has strong resistance to drought, cold, high temperature, and saline-alkali. The conserved domain FAE1-CUT1-RPPA and ACP-syn-III_C of the KCS gene family were used to search the KCS sequences across the whole genomic sequence of yellow horn; a total of 20 XsKCS genes were identified and divided into four subfamilies. The conserved motif and transmembrane structure analysis revealed that most XsKCSs had a conserved transmembrane domain except XsKCS10 and XsKCS20. The prediction of cis-acting elements of XsKCS genes showed that XsKCS genes contained many stress and hormone response elements, such as ABRE, MBS, and LTR. Furthermore, XsKCS genes exhibited differential expression profiles under abiotic stress and stress-related hormone treatment conditions. Transcriptomic data showed that XsKCS1, XsKCS11, and XsKCS17 had higher expression in yellow horn with high leaf cuticular wax, indicating that they may function in the cuticular wax accumulation and drought response. This study laid a foundation for further functional verification of XsKCS genes in yellow horn stress response.

Ontogenetic Variation in Macrocyclic and Hemicyclic Poplar Rust Fungi.

Melampsora larici-populina (Mlp), M. medusae (Mmed), M. magnusiana (Mmag), and M. pruinosae (Mpr) are epidemic rust fungi in China. The first two are macrocyclic rust fungi distributed in temperate humid environments. The latter two are hemicyclic rusts, mainly distributed in arid and semi-arid areas. Ontogenetic variation that comes with this arid-resistance is of great interest-and may help us predict the influence of a warmer, drier, climate on fungal phylogeny. To compare the differences in the life history and ontogeny between the two types of rust, we cloned mating type genes, STE3.4 and STE3.3 using RACE-smart technology. Protein structures, functions, and mutant loci were compared across each species. We also used microscopy to compare visible cytological differences at each life stage for the fungal species, looking for variation in structure and developmental timing. Quantitative PCR technology was used to check the expression of nuclear fusion and division genes downstream of STE3.3 and STE3.4. Encoding amino acids of STE3.3 and STE3.4 in hemicyclic rusts are shorter than these in the macrocyclic rusts. Both STE3.3 and STE3.4 interact with a protein kinase superfamily member EGG12818 and an E3 ubiquitin protein ligase EGG09709 directly, and activating G-beta conformational changes. The mutation at site 74th amino acid in the conserved transmembrane domain of STE3.3 ascribes to a positive selection, in which alanine (Ala) is changed to phenylalanine (Phe) in hemicyclic rusts, and a mutation with Tyr lost at site 387th in STE3.4, where it is the binding site for β-D-Glucan. These mutants are speculated corresponding to the insensitivity of hemicyclic rust pheromone receptors to interact with MFa pheromones, and lead to Mnd1 unexpressed in teliospora, and they result in the diploid nuclei division failure and the sexual stage missing in the life cycle. A Phylogenic tree based on STE3.4 gene suggests these two rust types diverged about 14.36 million years ago. Although these rusts share a similar uredia and telia stage, they show markedly different wintering strategies. Hemicyclic rusts overwinter in the poplar buds endophytically, their urediniospores developing thicker cell walls. They form haustoria with a collar-like extrahaustorial membrane neck and induce host thickened callose cell walls, all ontogenetic adaptations to arid environments.

A computational study of the structure and function of human Zrt and Irt-like proteins metal transporters: An elevator-type transport mechanism predicted by AlphaFold2.

The ZIP (Zrt and Irt-like proteins) protein family includes transporters responsible for the translocation of zinc and other transition metals, such as iron and cadmium, between the extracellular space (or the lumen of organelles) and the cytoplasm. This protein family is present at all the phylogenetic levels, including bacteria, fungi, plants, insects, and mammals. ZIP proteins are responsible for the homeostasis of metals essential for the cell physiology. The human ZIP family consists of fourteen members (hZIP1-hZIP14), divided into four subfamilies: LIV-1, containing nine hZIPs, the subfamily I, with only one member, the subfamily II, which includes three members and the subfamily gufA, which has only one member. Apart from the extracellular domain, typical of the LIV-1 subfamily, the highly conserved transmembrane domain, containing the binuclear metal center (BMC), and the histidine-rich intracellular loop are the common features characterizing the ZIP family. Here is presented a computational study of the structure and function of human ZIP family members. Multiple sequence alignment and structural models were obtained for the 14 hZIP members. Moreover, a full-length three-dimensional model of the hZIP4-homodimer complex was also produced. Different conformations of the representative hZIP transporters were obtained through a modified version of the AlphaFold2 algorithm. The inward and outward-facing conformations obtained suggest that the hZIP proteins function with an “elevator-type” mechanism.

The expression profile of a multi-stress inducible transient receptor potential vanilloid 4 (TRPV4) in Pacific oyster Crassostrea gigas

Transient receptor potential vanilloid 4 (TRPV4) is one of the major non-selective cation channel proteins, which plays a crucial role in sensing biotic and abiotic stresses, such as pathogen infection, temperature, mechanical pressure and osmotic pressure changes by regulating Ca2+ homeostasis. In the present study, a TRPV4 homologue was identified in Pacific oyster Crassostrea gigas, designated as CgTRPV4. The open reading frame (ORF) of CgTRPV4 was of 2298 bp encoding a putative polypeptide of 765 amino acid residues with three typical ankyrin domains and six conserved transmembrane domains of TRPV4 subfamily proteins, as well as multiple N-glycosylation sites, cAMP- and cGMP-dependent protein kinase phosphorylation sites, protein kinase C phosphorylation sites, casein kinase II phosphorylation sites, and prokaryotic membrane lipoprotein lipid attachment site. The deduced amino acid sequence of CgTRPV4 shared 20.5%-26.2% similarity with TRPV4s from other species. During the early ontogenesis stages of oyster, the mRNA transcripts of CgTRPV4 were detectable in all the stages with the highest expression level in fertilized eggs and the lowest in D-hinged larvae. In adult oyster, the CgTRPV4 mRNA could be detected in all the examined tissues, including gill, hepatopancreas, adductor muscle, labial palp, mantle and haemocyte, with the highest expression level in gill (45.08-fold of that in hepatopancreas, p < 0.05). In immunocytochemical assay, the CgTRPV4 positive signals were distributed in both endoplasmic reticulum and cytoplasmic membrane of oyster haemocytes. The mRNA expression of CgTRPV4 in gill was significantly up-regulated after high temperature stress at 30°C (p < 0.05) and after Vibrio splendidus stimulation (p < 0.05). These results indicated that CgTRPV4 was a classical member of TRPV4 family in oyster, which was induced by either biotic or abiotic stimulations and involved in mediating the stress response of oysters.

Molecular interaction of nitrate transporter proteins with recombinant glycinebetaine results in efficient nitrate uptake in the cyanobacterium Anabaena PCC 7120.

Nitrate transport in cyanobacteria is mediated by ABC-transporter, which consists of a highly conserved ATP binding cassette (ABC) and a less conserved transmembrane domain (TMD). Under salt stress, recombinant glycinebetaine (GB) not only protected the rate of nitrate transport in transgenic Anabaena PCC 7120, rather stimulated the rate by interacting with the ABC-transporter proteins. In silico analyses revealed that nrtA protein consisted of 427 amino acids, the majority of which were hydrophobic and contained a Tat (twin-arginine translocation) signal profile of 34 amino acids (1–34). The nrtC subunit of 657 amino acids contained two hydrophobic distinct domains; the N-terminal (5–228 amino acids), which was 59% identical to nrtD (the ATP-binding subunit) and the C-terminal (268–591), 28.2% identical to nrtA, suggesting C-terminal as a solute binding domain and N-terminal as ATP binding domain. Subunit nrtD consisted of 277 amino acids and its N-terminal (21–254) was an ATP binding motif. Phylogenetic analysis revealed that nitrate-ABC-transporter proteins are highly conserved among the cyanobacterial species, though variation existed in sequences resulting in several subclades. Nostoc PCC 7120 was very close to Anabaena variabilis ATCC 29413, Anabaena sp. 4–3 and Anabaena sp. CA = ATCC 33047. On the other, Nostoc spp. NIES-3756 and PCC 7524 were often found in the same subclade suggesting more work before referring it to Anabaena PCC 7120 or Nostoc PCC 7120. The molecular interaction of nitrate with nrtA was hydrophilic, while hydrophobic with nrtC and nrtD. GB interaction with nrtACD was hydrophobic and showed higher affinity compared to nitrate.

Genome-Wide Identification, Structure Characterization, Expression Pattern Profiling, and Substrate Specificity of the Metal Tolerance Protein Family in Canavalia rosea (Sw.) DC.

Plant metal tolerance proteins (MTPs) play key roles in heavy metal absorption and homeostasis in plants. By using genome-wide and phylogenetic approaches, the origin and diversification of MTPs from Canavalia rosea (Sw.) DC. was explored. Canavalia rosea (bay bean) is an extremophile halophyte with strong adaptability to seawater and drought and thereby shows specific metal tolerance with a potential phytoremediation ability. However, MTP genes in leguminous plants remain poorly understood. In our study, a total of 12 MTP genes were identified in C. rosea. Multiple sequence alignments showed that all CrMTP proteins possessed the conserved transmembrane domains (TM1 to TM6) and could be classified into three subfamilies: Zn-CDFs (five members), Fe/Zn-CDFs (five members), and Mn-CDFs (two members). Promoter cis-acting element analyses revealed that a distinct number and composition of heavy metal regulated elements and other stress-responsive elements existed in different promoter regions of CrMTPs. Analysis of transcriptome data revealed organ-specific expression of CrMTP genes and the involvement of this family in heavy metal stress responses and adaptation of C. rosea to extreme coral reef environments. Furthermore, the metal-specific activity of several functionally unknown CrMTPs was investigated in yeast. These results will contribute to uncovering the potential functions and molecular mechanisms of heavy metal absorption, translocation, and accumulation in C. rosea plants.

Functional characterization of two ecto-nucleoside triphosphate diphosphohydrolase 2 genes in Japanese flounder (Paralichthys olivaceus) head kidney macrophages

Ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases) are pivotal regulators of extracellular ATP-mediated purinergic immune signaling. ENTPDase2 is a member of the cell surface-bound ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) protein family that hydrolyzes extracellular nucleoside 5′-triphosphates and nucleoside 5′-diphosphates. However, the immune relevance of ENTPDase2 in fish has not been elucidated. In the present study, from a comparative immunological perspective, we functionally characterized two ENTPDase2 transcript variants (namely ENTPDase2 and ENTPDase2a) from Japanese flounder (Paralichthys olivaceus). Sequence analysis indicates that the deduced Japanese flounder ENTPDase2 and ENTPDase2a proteins possess two conserved transmembrane domains and five apyrase conserved regions that are present in ENTPDase family proteins. However, these proteins only share 54% amino acid sequence identity. Tissue expression analysis revealed that both ENTPDase2 and ENTPDase2a mRNA transcripts are ubiquitously expressed in all examined Japanese flounder tissues, whereas ENTPDase2 is dominantly expressed in blood and ENTPDase2a is abundantly expressed in muscle. Immune challenge experiments showed that ENTPDase2 and ENTPDase2a were significantly upregulated by both inflammatory stimulation and Edwardsiella tarda infection. In addition, the expression of ENTPDase2 and ENTPDase2a was modulated by extracellular ATP (eATP) stimulation in a dose-dependent manner. Furthermore, immunolocalization and functional studies demonstrated that both ENTPDase2 and ENTPDase2a are functional glycosylated plasma membrane proteins. However, ENTPDase2a exhibits greater activity in the hydrolysis of eATP than ENTPDase2 and ENTPDase1 proteins. Finally, knockdown of the ENTPDase2 gene by small interfering RNA significantly upregulated the expression of eATP-induced proinflammatory cytokines IL-1beta, TNF-alpha and G-CSF in Japanese flounder head kidney macrophages, while knockdown of ENTPDase2a only upregulated eATP-induced IL-1beta expression. Taken together, our findings suggest that the two functional Japanese flounder ENTPDase2 isoforms play an essential role in the downregulation of eATP-induced proinflammatory cytokine expression in fish by degrading the available ATP levels in the extracellular milieu.

Extramembranous Regions in G Protein-Coupled Receptors: Cinderella in Receptor Biology?

G protein-coupled receptors (GPCRs) are the largest class of membrane proteins involved in signal transduction and are characterized by seven transmembrane domain architecture interconnected by extra- and intracellular loops. These loops, along with the N- and C-terminal domains, constitute the extramembranous regions in GPCRs. These regions, accounting for ~ 40% or more amino acid residues across different GPCR classes, are distinct from the conserved transmembrane domains in terms of nonconservation of sequence, diversity in length, and conformational heterogeneity. Due to technical challenges in exploring the molecular basis underlying the relation between structure, dynamics, and function in these regions, their contribution to GPCR organization and signaling remain underappreciated. Despite existingliterature on the involvement of GPCR loops in numerous aspects of GPCR biology, the functional relevance of GPCR loops in the context of their inherent conformational heterogeneity and probable membrane interaction are not well understood. This review focuses on highlighting these aspects of GPCR extramembranous regions in the overall context of GPCR organization, dynamics, and biology. We envision that a judicious combination of insights obtained from structured transmembrane domains and disordered extramembranous regions in GPCRs would be crucial in arriving at a comprehensive understanding of GPCR structure, function, and dynamics, thereby leading to efficient drug discovery.

The lipid flippase subunit Cdc50 is required for antifungal drug resistance, endocytosis, hyphal development and virulence in Candida albicans.

Cdc50 is the non-catalytic subunit of the flippase that establishes phospholipid asymmetry in membranes and functions in vesicle-mediated trafficking in Saccharomyces cerevisiae. Here, we have identified the homologous gene CaCDC50 that encodes a protein of 396 amino acids with two conserved transmembrane domains in Candidaalbicans. Deletion of CaCDC50 results in C. albicans cells becoming sensitive to the antifungal drugs azoles, terbinafine and caspofungin, as well as to the membrane-perturbing agent sodium dodecyl sulfate. We also show that CaCDC50 is involved in both endocytosis and vacuolar function. CaCDC50 confers tolerance to high concentrations of cations, although it is not required for osmolar response. Moreover, deletion of CaCDC50 leads to severe defects in hyphal development of C. albicans cells and highly attenuated virulence in the mouse model of systemic infection. Therefore, CaCDC50 regulates cellular responses to antifungal drugs, cell membrane stress, endocytosis, filamentation and virulence in the human fungal pathogen C. albicans.

Characterization of the Gap Junction Gene Innexin in Lymnaea stagnalis

Connections between neurons called synapses are key components underlying the basic functioning of the nervous system. From simple reflexes to high cognitive functions, all nervous system operations rely on two main forms of synaptic communication to efficiently transmit signals: chemical (transmitter‐mediated) and electrical (gap junction protein‐mediated). Emerging research has proved the widespread expression of gap junction (GJ) channels in many species including humans and in a variety of brain regions, and recent studies have indicated that GJs play vital roles in the early development of brains; yet, little is known about the regulatory factors (both intrinsic and extrinsic) and mechanisms that control the development of electrical synapses. The current lack of fundamental knowledge in the area of electrical synaptogenesis and regulation is mainly attributed to the extreme complexity of the mammalian brain which does not permit long‐term, real‐time neuronal recordings throughout electrical synapse formation. Therefore, we opt to use an in vitro experimental system, the mollusc Lymnaea stagnalis, in which physically large, well‐defined neuronal networks and a published genome allow unique opportunities to study cell‐specific expression of genes throughout electrical synapse formation in the developing brain. The objective of this study was to characterize the expression, localization, and regulation of the GJ‐encoding gene innexin in L. stagnalis during development. To do this, the central nervous system (CNS) of L. stagnalis was dissected and underwent either subsequent RNA extraction (for cloning) or single‐cell extraction. Innexin was cloned utilizing degenerate primers and 3′ and 5′ rapid amplification of cDNA ends (RACE) systems, and, from the innexin sequence information, primers were designed for RT‐ and qPCR. The innexin cloning experiments revealed 10 isoforms of the gene in L. stagnalis, all with conserved transmembrane domain regions. To elucidate the tissue‐ and cell‐specific expression of innexin isoforms, RT‐PCR was performed on various L. stagnalis organs, and qPCR was performed on cells in the CNS known to form electrical synapses. Innexin isoforms are expressed ubiquitously throughout L. stagnalis but vary between individuals and across developmental time. In situ hybridization also demonstrated variable localization of one innexin isoform across the CNS, with only some electrical synapse‐forming cells localizing the isoform mRNA on the plasma membrane and others not expressing the mRNA at all. Extrinsic cellular molecules called trophic factors were found to regulate electrical synapse formation and innexin expression in vitro. Overall, the GJ gene innexin has variable expression and distribution throughout the life of L. stagnalis, indicating the critical role the gene plays in the proper development of invertebrates and, by conserved functional homology, vertebrates.Support or Funding InformationThis project was supported by the Saint Louis University Start‐up Fund, the President Research Fund, the Beaumont Faculty Development Fund, and Spark Microgrant awarded to Dr. Xu. This project was also supported by the Sigma Xi Grant‐In‐Aid of Research awarded to Ms. Brittany Mersman.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Role of YlBud8 in the Regulation of Cell Separation in the Yeast Yarrowia lipolytica.

The spatial landmark protein Bud8 plays a crucial role in bipolar budding in the budding yeast Saccharomyces cerevisiae. The unconventional yeast Yarrowia lipolytica can also bud in a bipolar pattern, but is evolutionarily distant from S. cerevisiae. It encodes the protein YALI0F12738p, which shares the highest amino acid sequence homology with S. cerevisiae Bud8, sharing a conserved transmembrane domain at the C-terminus. Therefore, we named it YlBud8. Deletion of YlBud8 in Y. lipolytica causes cellular separation defects, resulting in budded cells remaining linked with one another as cell chains or multiple buds from a single cell, which suggests that YlBud8 may play an important role in cell separation, which is distinct from the function of Bud8 in S. cerevisiae. We also show that the YlBud8-GFP fusion protein is located at the cell membrane and enriched in the bud cortex, which would be consistent with a role in the regulation of cell separation. The coiled-coil domain at the N-terminus of YlBud8 is important to the correct localization and function of YlBud8, as truncated proteins that do not contain the coiled-coil domain cannot rescue the defects observed in Ylbud8Δ. This finding suggests that a new signaling pathway controlled by YlBud8 via regulation of cell separation may exist in Y. lipolytica.

Molecular characterization and expression analysis of Asian swamp eel (Monopterus albus) CXC chemokine receptor (CXCR) 1a, CXCR1b, CXCR2, CXCR3a, CXCR3b, and CXCR4 after bacteria and poly I:C challenge

The CXC chemokine receptors (CXCRs) play critical roles in innate and adaptive immune systems. In this study, six Asian swamp eel (Monopterus albus) CXCRs (MaCXCR1–4) were identified and their molecular characterization and expression patterns were analyzed. The open reading frames (ORFs) of MaCXCR1a, MaCXCR1b, MaCXCR2, MaCXCR3a, MaCXCR3b, and MaCXCR4 were 1074 bp (base pairs), 1080 bp, 1125 bp, 1146 bp, 1083 bp, and 1140 bp, and encoded proteins of 357 aa (amino acids), 359 aa, 374 aa, 381 aa, 360 aa, and 379 aa, respectively. All these CXCRs have seven conserved transmembrane domains and four cysteines (with the exception of MaCXCR3b). Multiple sequence alignment revealed that the MaCXCRs possess a typical G-protein receptor family 1 signature and a DRY motif. There are also one to four potential N-glycosylation sites in the extracellular regions of the MaCXCRs, mainly distributed in the N-terminus and extracellular hydrophilic loop (ECL) 2 region. Phylogenetic analysis demonstrated that the MaCXCRs were clustered together with homologous proteins from other fish. Taken together with the amino acid identity and similarity analysis, these results suggested that the MaCXCRs are conserved with other homologous genes, in which CXCR4 is more conserved than CXCR1–3. The MaCXCRs loci showed conserved synteny among teleost fish, and we found that human CXCR1 shares a common ancestor with fish CXCR1a. MaCXCRs were constitutively expressed in a wide range of tissues (especially in immune-related tissues) with different expression levels, suggesting that the MaCXCRs have different roles in un-stimulated tissues, and may play vital roles under normal conditions. MaCXCRs showed different fold changes in the spleen after Aeromonas veronii and polyinosinic-polycytidylic acid (poly I:C) challenge, which suggested that MaCXCR1a and MaCXCR3a have longer antiviral activities compared with their antibacterial functions, and that MaCXCR1b possesses stronger antiviral than antibacterial activity. MaCXCR4 may play vital roles during bacterial and viral infection; however, MaCXCR2 has relatively small effect in antibacterial and antiviral responses. The differential responses of these genes to bacteria and poly I:C implied the differences in the mechanisms of defense against viruses and bacteria.

Genome-wide Identification, Classification and Expression Analysis of the Mildew Resistance Locus O ( MLO ) Gene Family in Sweet Orange ( Citrus sinensis )

ABSTRACT Mildew resistance Locus O (MLO), a gene family specific to plants, plays significant roles in the resistance to powdery mildew (PM) and response to a variety of abiotic stresses, plant growth and development. Despite their importance as barley, rice, wheat, few studies are reported in dicots except Arabidopsis; no global analysis has been performed in the burgeoning model fruit plant sweet orange (Citrus sinensis). The recent release of the genome sequences of C. sinensis provides an opportunity to conduct a comprehensive overview the evolution and features of the MLO gene family in sweet orange. In this study, amount to 14 members of the Citrus sinensis MLO gene (CisMLO) family according to their gene structures, conserved motifs, and similitude among their presumptive Arabidopsis and rice orthologs were identified in silico. Based on these analyses, all CisMLOs were grouped into six clades and expanded partly due to one tandem duplication and two segmental duplication events. Survey of their chromosomal distributions uncovered that 14 CisMLOs are localized across 6 chromosomes. Multiple-sequence alignments showed that 11 of them shared seven highly conserved transmembrane domains (TMs), while all of the sweet orange MLO proteins except CisMLO4/14 had a calmodulin-binding domain for MLO function. Expression analysis demonstrated that the MLO gene family has a diverse tissue-specific expression profiles in the sweet orange development and plays potential critical roles in stress responses. These findings will facilitate further studies of evolutionary pattern and biological functions of MLO genes in sweet orange.

A point mutation in the ion conduction pore of AMPA receptor GRIA3 causes dramatically perturbed sleep patterns as well as intellectual disability.

The discovery of genetic variants influencing sleep patterns can shed light on the physiological processes underlying sleep. As part of a large clinical sequencing project, WGS500, we sequenced a family in which the two male children had severe developmental delay and a dramatically disturbed sleep-wake cycle, with very long wake and sleep durations, reaching up to 106-h awake and 48-h asleep. The most likely causal variant identified was a novel missense variant in the X-linked GRIA3 gene, which has been implicated in intellectual disability. GRIA3 encodes GluA3, a subunit of AMPA-type ionotropic glutamate receptors (AMPARs). The mutation (A653T) falls within the highly conserved transmembrane domain of the ion channel gate, immediately adjacent to the analogous residue in the Grid2 (glutamate receptor) gene, which is mutated in the mouse neurobehavioral mutant, Lurcher. In vitro, the GRIA3(A653T) mutation stabilizes the channel in a closed conformation, in contrast to Lurcher. We introduced the orthologous mutation into a mouse strain by CRISPR-Cas9 mutagenesis and found that hemizygous mutants displayed significant differences in the structure of their activity and sleep compared to wild-type littermates. Typically, mice are polyphasic, exhibiting multiple sleep bouts of sleep several minutes long within a 24-h period. The Gria3A653T mouse showed significantly fewer brief bouts of activity and sleep than the wild-types. Furthermore, Gria3A653T mice showed enhanced period lengthening under constant light compared to wild-type mice, suggesting an increased sensitivity to light. Our results suggest a role for GluA3 channel activity in the regulation of sleep behavior in both mice and humans.

Dopamine Transporter Amino and Carboxyl Termini Synergistically Contribute to Substrate and Inhibitor Affinities

Extracellular dopamine and serotonin concentrations are determined by the presynaptic dopamine (DAT) and serotonin (SERT) transporters, respectively. Numerous studies have investigated the DAT and SERT structural elements contributing to inhibitor and substrate binding. To date, crystallographic studies have focused on conserved transmembrane domains, where multiple substrate binding and translocation features are conserved. However, it is unknown what, if any, role the highly divergent intracellular N and C termini contribute to these processes. Here, we used chimeric proteins to test whether DAT and SERT N and C termini contribute to transporter substrate and inhibitor affinities. Replacing the DAT N terminus with that of SERT had no effect on DA transport Vmax but significantly decreased DAT substrate affinities for DA and amphetamine. Similar losses in uptake inhibition were observed for small DAT inhibitors, whereas substituting the DAT C terminus with that of SERT affected neither substrate nor inhibitor affinities. In contrast, the N-terminal substitution was completely tolerated by the larger DAT inhibitors, which exhibited no loss in apparent affinity. Remarkably, all affinity losses were rescued in DAT chimeras encoding both SERT N and C termini. The sensitivity to amino-terminal substitution was specific for DAT, because replacing the SERT N and/or C termini affected neither substrate nor inhibitor affinities. Taken together, these findings provide compelling experimental evidence that DAT N and C termini synergistically contribute to substrate and inhibitor affinities.